Light-triggered ruthenium-catalyzed allylcarbamate cleavage in biological environments

Article abstract of DOI:10.1021/om3001668

The sandwich complex [Cp Ru(η⁶-pyrene)]PF₆ (Cp* = η⁵-C₅(CH₃)₅) serves as a photoactivatable catalyst for the conversion of N-allylcarbamates into their amines in the presence of thiophenol under biorelevant conditions (water, air, plus aliphatic thiols) and even in mammalian cells. This new phototriggered substrate/catalyst pair points towards applications in chemical biology and medicinal chemistry where signal amplification is combined with spacial and temporal control.

Full text of DOI:10.1021/om3001668

Note
pubs.acs.org/Organometallics
Light-Triggered Ruthenium-Catalyzed Allylcarbamate Cleavage in
Biological Environments
Pijus K. Sasmal,† Susana Carregal-Romero,‡ Wolfgang J. Parak,‡ and Eric Meggers*,†,§
†Fachbereich Chemie, Philipps-Universitat Marburg, Hans-Meerwein-Strasse, 35043 Marburg, Germany
̈
‡Fachbereich Physik and WZMW, Philipps-Universitat Marburg, Renthof 7, 35037 Marburg, Germany
̈
§Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's
Republic of China
ABSTRACT: The sandwich complex [Cp*Ru(η6-pyrene)]-
PF6 (Cp* = η5-C5(CH3)5) serves as a photoactivatable catalyst
for the conversion of N-allylcarbamates into their amines in
the presence of thiophenol under biorelevant conditions
(water, air, plus aliphatic thiols) and even in mammalian cells.
This new phototriggered substrate/catalyst pair points towards
applications in chemical biology and medicinal chemistry
where signal amplification is combined with spacial and
temporal control.
he modulation, sensing, and imaging of biological
processes in biomedical research typically relies on
under air (Scheme 1).11 It is noteworthy that no products were
formed in the absence of UV light.
T
stoichiometric events, whereas the catalytic initiation of
biological processes provides signal amplification, for example,
by turning over caged substrates multiple times, by catalytically
labeling or deactivating target biomolecules, and by catalytically
activating prodrugs.1 This advantage of catalytic turnover has
been successfully exploited in the area of enzyme-triggered
bioimaging.2 However, the design of bioorthogonal synthetic
catalyst/substrate pairs which can passively diffuse into cells,
such as a typical tool compound used in chemical biology
studies, is a highly formidable challenge and no useful systems
exist to date.3−5 We recently reported that the organometallic
half-sandwich complex [Cp*Ru(COD)Cl] (1; COD = 1,5-
cyclooctadiene) can catalyze the conversion of N-allylcarba-
mates into their amines in the presence of thiols, water, and
air.3 We here wish to disclose a photoactivated version of this
catalytic system, namely [Cp*Ru(η6-pyrene)]PF6 (2) (Scheme
1), which allows light-triggered catalysis with temporal and in
the future also spatial control.6,7
In order to test the photoinduced catalytic cleavage of
allylcarbamates under biologically more relevant conditions, we
used the previously developed bis-allyloxycarbonyl-modified
rhodamine 110 (5) (Scheme 2).3 This “caged” fluorophore is
nonfluorescent but can be converted to the highly fluorescent
rhodamine 110 (6) upon carbamate cleavage, thus allowing us
to monitor the photocatalysis by simple fluorescence readout.
Accordingly, upon UV photolysis, the caged rhodamine 5 in the
presence of β-mercaptoethanol, PhSH, and catalyst 2 (20 mol
%) resulted in the rapid formation of rhodamine 110 (6) with
maximum yields already reached after approximately 3 min
(Figure 1). The formation of rhodamine 110 correlated with
the formation of the fluorescent pyrene in the course of the
photoactivation of 2, as demonstrated in Figure 1. Further data
shown in Table 1 reveal that no significant amounts of
rhodamine 110 are formed without catalyst (Table 1, entry 1)
or in the absence of thiols (Table 1, entry 2). Obviously, thiols
are required for the Ru-catalyzed cleavage of the allylcarbamates
(Table 1, entries 3−6). In fact, the more nucleophilic
thiophenol (Table 1, entry 5) or combinations of aliphatic
and aromatic thiols (Table 1, entries 5 and 6) work significantly
better than aliphatic thiols alone (Table 1, entries 3 and 4).
Finally, we investigated the photoactivated allylcarbamate
cleavage in mammalian cells. For this, cultured HeLa cells were
first incubated with the caged fluorophore 5 (100 μM),
followed by a washing step, so that the caged fluorophore 5 was
only located within the HeLa cells but not in the culture
RESULTS AND DISCUSSION
■
The sandwich complex [Cp*Ru(η6-pyrene)]PF6 was first
reported by Mann et al. and was demonstrated to be quite
stable toward thermal replacement of the pyrene moiety.8,9 On
the basis of the known photochemical lability of Ru(η6-arene)
sandwich coordination modes,10 we envisioned that the
released solvent-coordinated fragment [Cp*Ru(solvent)3]+
should be capable of serving as a catalyst for a variety of
transformations. Indeed, we found that the complex 2 (10 mol
%), upon irradiation with λ ≥330 nm for 5 min, catalyzes the
cleavage of a representative selection of allylcarbamates 3a−d
to their respective amines 4a−d in good yields of 70−83% in
the presence of 5 equiv of thiophenol at room temperature and
Special Issue: Organometallics in Biology and Medicine
Received: February 28, 2012
Published: May 9, 2012
© 2012 American Chemical Society
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Organometallics
Note
Scheme 1. Previous Thermal and New Photoactivated
Ruthenium Catalysts (λ ≥ 330 nm) for the Cleavage of
b
Allylcarbamates under Biorelevant Conditions
Figure 1. Fluorescence-monitored formation of rhodamine 110 and
pyrene in the photoinduced catalytic conversion 5 → 6 in the presence
of 20 mol % [Cp*Ru(η6-pyrene)]PF6. Reaction conditions: caged
rhodamine 5 (0.5 mM) in DMSO/H2O (1/1) with β-mercaptoetha-
nol (5 mM), PhSH (5 mM), and catalyst 2 (0.1 mM) under photolysis
with λ ≥330 nm for the monitored reaction time. Yields were
determined by fluorescence measurements (pyrene, λex 319 nm, λem
390 nm; rhodamine 110, λex 488 nm, λem 520 nm), the data were fitted
to an exponential equation, and standard deviations from three
independent experiments are given.
Table 1. Light-Induced Catalytic Cleavage of
Bisallyloxycarbonylrhodamine 5 with [Cp*Ru(η6-
pyrene)]PF6 under Biologically Relevant Conditions
a
a
b
Isolated as BOC-protected amine. The yields for the thermal
b
entry
amt of cat. (mol %)
thiol
yield (%)
reaction are shown in parentheses for comparison and are taken from
ref 3.
1
2
3
4
5
6
none
20
ME
0.5 0.1
1.2 0.3
none
20
ME
13
14
93
90
2
2
4
3
Scheme 2. N,N-Bisallyloxycarbonylrhodamine 110 (5) as a
“Caged” Fluorophore
20
Cys
20
PhSH
ME + PhSH
20
a
General reaction conditions: caged rhodamine 5 (0.5 mM) and
catalyst 2 (0.1 mM) in 1/1 DMSO/sodium phosphate buffer (1 mM,
pH 7.25) at 37 °C for 10 min under irradiation at λ ≥330 nm. The
concentrations of β-mercaptoethanol (ME), PhSH, and cysteine (Cys)
b
were all 5 mM. Yields were determined by fluorescence measure-
ments, and the standard deviations from three independent experi-
ments are provided.
medium. At this stage, no rhodamine 110 fluorescence could be
detected (Figure 2A), after the following addition of either
complex 2 alone or complex 2 together with thiophenol
(Figures 2B,C). However, the addition of complex 2 followed
by a 10 min irradiation led to a small increase in fluorescence
(6-fold) (Figure 2D), whereas a much stronger fluorescence
increase was observed upon photolysis in the presence of
complex 2 together with thiophenol: an irradiation time of 5
min resulted in an increase in the overall fluorescence intensity
by 35-fold (Figure 2E), whereas an irradiation time of 10 min
increased the fluorescence intensity further to overall 70-fold
(Figure 2F).12 This benefit of thiophenol is consistent with our
model experiments shown in Table 1. Furthermore, the
distribution of fluorescence within the cellular cytosol implies
that the ruthenium sandwich complex is membrane-perme-
able.13,14
living mammalian cells. This phototriggered substrate/catalyst
pair might become an attractive tool for future applications that
require spatial and temporal control.
EXPERIMENTAL SECTION
■
Compound Synthesis. Allylcarbamates 3a−d,3,15 [Cp*Ru-
(pyrene)]PF6 (2),8 and N,N-bisallyloxycarbonylrhodamine 110 (5)3
were synthesized according to published procedures.
Photoinduced Cleavage of N-Allyloxycarbonylanilines. A
representative example is given: 4-chloro-N-allyloxycarbonylaniline
(15 mg, 0.071 mmol), [Cp*Ru(pyrene)]PF6 (4 mg, 0.007 mmol, 10
mol %), and thiophenol (36 μL, 0.35 mmol) were dissolved in DMSO
(105 μL), and the solution was irradiated for 5 min with a 200 W
Hg(Xe) arc lamp (Newport) in combination with a λ ≥330 nm filter.
Afterward, the reaction mixture was stirred overnight at room
temperature in air. The resulting solution was diluted 10-fold with
water, extracted with Et2O (5 × 5 mL), dried over Na2SO4,
concentrated in vacuo, and purified by silica gel flash column
chromatography with hexane/ethyl acetate (5/1 to 3/1) to afford
7.8 mg (83%) of 4-chloroaniline. Analogous reactions have been
performed under thermal conditions with [Cp*Ru(COD)Cl].3
Photoinduced Cleavage of Caged Rhodamine 5. Biscarba-
mate 5 (0.5 mM) and ruthenium complex 2 (0.1 mM) in 1/1 DMSO/
CONCLUSION
■
We here demonstrated that, upon photoactivation, [Cp*Ru(η6-
pyrene)]PF6 is capable of catalyzing the conversion of N-
allylcarbamates to their respective amines in the presence of
thiophenol under biologically relevant conditions and even in
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Note
catalyst concentration of 20 μM. After moderate agitation for a few
seconds, the standard-bottom dishes were exposed to light of λ ≥330
nm for 5 min. Afterward, the medium was removed and washed one
time with PBS buffer. Then, the cells were fixed and permeabilized
with cold methanol for 5 min at −20 °C. Subsequently, methanol was
removed and dishes containing cells were stored at −20 °C until
fluorescence images were recorded with a LSM 510 META confocal
microscope (Zeiss). Images comprised phase contrast and green
fluorescence channels. The image analysis was performed with the
software Image J as reported recently.16
AUTHOR INFORMATION
Corresponding Author
*E-mail: meggers@chemie.uni-marburg.de.
■
Notes
The authors declare no competing financial interest.
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■
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Figure 2. Confocal fluorescence imaging of [Cp*Ru(pyrene)]PF6-
induced uncaging of the bisallylcarbamate-protected rhodamine 110
(5) inside HeLa cells. HeLa cells were incubated with caged
rhodamine 5 (100 μM) for 25 min and then washed with PBS buffer.
(A) after the washing step; (B) after the addition of ruthenium
complex 2 (20 μM) and a 10 min incubation time; (C) after the
addition of ruthenium complex 2 (20 μM) and thiophenol (1 mM)
and a 10 min incubation time without photolysis; (D) ruthenium
complex 2 (20 μM) added and photolyzed for 10 min with λ ≥330
nm; (E) ruthenium complex 2 (20 μM) and thiophenol (1 mM)
added and photolyzed for 5 min with λ ≥330 nm; (F) ruthenium
complex 2 (20 μM) and thiophenol (1 mM) added and photolyzed for
10 min with λ ≥330 nm.
sodium phosphate buffer (1 mM, pH 7.25) were irradiated with λ
≥330 nm at 37 °C for 10 min in the absence or presence of additional
thiols (5 mM each). Yields were determined by measurement of the
generated fluorescence of rhodamine 110 with a SpectraMax M5
fluorescence plate reader from Molecular Devices (λex 488 nm, cutoff
495 nm, λem 520 nm) and comparison with a calibration curve
prepared with rhodamine 110 (Table 1).3
Fluorescence Confocal Imaging of Mammalian Cells. HeLa
cells were cultured in Dulbecco’s modified Eagle medium (DMEM,
Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS,
Biochrom), glutamine (2 mM), and penicillin/streptomycin (100 μg/
mL). One day in advance of the experiments, approximately 1 × 105
cells were plated on standard-bottom dishes (35/12/1.5 mm) in media
(2 mL). Rhodamine biscarbamate 5 in DMSO (10 μL, 20 mM stock)
was added to the standard-bottom dishes containing HeLa cells and
briefly agitated to reach a homogeneous concentration of 100 μM.
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and cells were washed successively with PBS buffer (2 × 1 mL) and
replaced by 1 mL of fresh medium. Then, [Cp*Ru(η6-pyrene)]PF6 (4
μL of a 5 mM stock solution in DMSO) was added, resulting in a
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